This invention relates to the Lepidopteran resistant transgenic variety of corn (Zea mays) plant referred to herein as event MON89034, and to unique DNA sequences present that, when detected in any sample or variety of corn, are diagnostic for the presence of the transgenic corn plant event MON89034 in that sample or variety, and also relates to the detection of the transgene/genomic insertion region in corn MON89034, and progeny plants and seeds derived therefrom.
The corn plant event MON89034 is particularly resistant to insects in the Lepidoptera family such as Fall armyworm (Spodoptera frugiperda), European corn borer (Ostrinia nubilalis), corn earworm (Helicoverpa zea), southwestern corn borer (Diatraea grandiosella), and black cutworm (Agrotis ipsilon) and the like, all of which are agronomically important insect pests.
Corn is an important crop and is a primary food source in many areas of the world. Biotechnology methods have been applied to corn for the purpose of improving agronomic traits and the quality of the product. One such agronomic trait is insect resistance, for example, genetically engineered resistance to lepidopteran and coleopteran species that arises in corn plants genetically engineered to contain one or more genes encoding insecticidal agents (see for example, U.S. Pat. No. 6,489,542 and U.S. Pat. No. 6,620,988). It is advantageous to detect the presence of a particular transgenic event in a biological sample in order to determine whether one or more progeny of a sexual cross contains the transgenic material. For example, the detection of the event in a sample is important for licensing purposes, for establishing and maintaining purity standards, important for complying with regulatory agencies, for complying with food ingredient standards, for use in legal proceedings in establishing that one or more particular individuals or entities has been using the particular event without a license from the owner or licensee of any patents directed to the transgenic event, and for insuring compliance with various government regulations and/or laws.
In addition, methods that enable the detection of a particular plant would be helpful when complying with regulations requiring the pre-market approval and labeling of foods derived from the recombinant crop plants. Individuals or entities that are resistant to the presence of a transgenic event in a sample also desire reliable methods for detecting the presence of the transgene in a sample in order for them to be able to capitalize on their business, which takes advantage of an absence of transgenes in their products.
Despite these advantages, it is possible that insects may evolve resistance to plants expressing only one B. thuringiensis δ-endotoxin. Such resistance, should it become widespread, would clearly limit the commercial value of germplasm containing single Bt genes.
One possible way of increasing the effectiveness of insecticidal agents provided via transgenic plants and directed at controlling target insect pests and contemporaneously reducing the likelihood of emergence of insect pests resistant to such insecticidal agents would be to ensure that transgenic crops express high levels of these insecticidal agents, such as Bacillus thuringiensis delta-endotoxins (McGaughey and Whalon (1992), Science 258:1451-55; Roush (1994) Biocontrol. Sci. Technol. 4:501-516). In addition, having a repository of insecticidal genes that are effective against groups of insect pests and which manifest their effects through different modes of action can safeguard against development of resistance. The onset of resistance could be substantially delayed as a result of providing a crop that expresses two or more insecticidal activities exhibiting overlapping toxicity to the same insect species. One means for achieving such dual modes of action could be to provide a plant expressing a Bt gene toxic to a particular insect species along with a dsRNA that is provided for the purpose of targeting for suppression an essential gene of the same insect species targeted by the Bt toxin, the dsRNA eliciting an RNAi response upon ingestion by the target pest, providing a means for redundancy in the event that the insect develops resistance either to the dsRNA or to the Bt gene. Alternatively, co-expression in a plant of two or more insecticidal toxins both toxic to the same insect species but each exhibiting a different mode of effectuating its killing activity, particularly when both are expressed at high levels, provides a means for effective resistance management. Examples of such insecticides useful in such combinations include but are not limited to Bt toxins, Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins, deallergenized and de-glycosylated patatin proteins and/or permuteins, plant lectins, and the like.
The expression of foreign genes in plants is known to be influenced by their chromosomal position, perhaps due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulation elements (e.g., enhancers) close to the integration site (Weising et al. (19880 Ann. Rev. Genet 22:421-477). For this reason, it is often necessary to screen a large number of events in order to identify an event characterized by optimal expression of an introduced gene of interest. Even then, with dozens or even hundreds of different transgenic events in hand, there is no certainty of success in identifying a single transgenic event that provides the optimum levels of expression of the at least two different toxins or insecticidal agents and lacks any undesirable agronomic deficiencies or phytotoxic effects, either as a result of the insertion into some essential or partially essential region of the plant genome, or as a result of toxic effects brought about by the levels of expression of the transgenes. For example, it has been observed in plants and in other organisms that there may be wide variation in the levels of expression of an introduced gene among events. There may also be differences in spatial or temporal patterns of expression, for example, differences in the relative expression of a transgene in various plant tissues, that may not correspond to the patterns expected from transcriptional regulatory elements present in the introduced gene construct. For this reason, it is common to produce several hundreds to several thousands different events and screen the events for a single event that has the desired transgene expression levels and patterns for commercial purposes. An event that has the desired levels or patterns of transgene expression is useful for introgressing the transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny of such crosses maintain the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in a number of varieties that are suitably adapted to specific local growing conditions.
It is possible to detect the presence of a transgene by any well known nucleic acid detection method such as the polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. These detection methods generally focus on frequently used genetic elements, such as promoters, terminators, marker genes, or even the coding sequence encoding the protein or dsRNA of interest expressed from the transgene(s), etc. As a result, such methods may not be useful for discriminating between different events, particularly those produced using the same DNA construct, unless the sequence of chromosomal DNA adjacent to the inserted DNA (“flanking DNA”) is known. Depending on the method used for introducing the transgene(s) into a plant genome, abberant or unusual effects can be observed, which often severly complicate the identification of the plant genome sequences flanking the transgenic DNA that was intended to be introduced into the plant. Often, rearrangements of the inserted DNA, rearrangements of the flanking genome DNA, or rearrangements of both the inserted DNA and the flanking genome DNA are prevalent, and complicate the analysis of the insertional event being evaluated. Therefore, it is advantageous to have a means for selecting, for identifying, and for insuring the purity and characteristics of a particular transgenic event in a sample, and the only way to accomplish this is to identify one or more unique sequences associated only with the desired transgenic event, and the presence of such sequences in a biological sample containing DNA of the plant species into which the transgenic DNA was inserted to give rise to the event are thus diagnostic for the event in such sample.